Many scientific questions and bioengineering goals relate to the monitoring and control of RNA functions in living cells. The ability to monitor and perturb RNA in living cells would benefit greatly from a way of systematically targeting unmodified RNA sequences for observation and control. Proteins that can bind arbitrary DNA sequences in a modular fashion, such that varying the sequence of building blocks in a given protein can result in essentially any DNA sequence being targeted [Buxbaum A R, Haimovich G, Singer R H (2014) In the right place at the right time: visualizing and understanding mRNA localization. Nat Rev Mol Cell Biol 16(2):95-109], are having much impact in the study and engineering of genomes. If a similar protein architecture could be devised for RNA sequences, so that any RNA sequence could be targeted simply by varying the sequence of building blocks within a designed protein, this could open up new abilities for the observation, control, and mapping of endogenous RNAs and their products.
A powerful strategy is to modify a target RNA by inserting an exogenous sequence like MS2 or PP7, so that the corresponding RNA binding protein can deliver a reporter or RNA modification enzyme to an RNA of interest [Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843-1846 (2011); Miller, M. T., Higgin, J. J. & Hall, T. M. T. Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p. Nature structural & molecular biology 15, 397-402 (2008); Wang, X., Zamore, P. D. & Hall, T. M. Crystal structure of a Pumilio homology domain. Mol Cell 7, 855-865 (2001)]. Ideally one could target unmodified RNA, both for simplicity and to preserve as much native RNA structure and function as possible [Re A, Joshi T, Kulberkyte E, Morris Q, Workman C T (2014) RNA-protein interactions: an overview. Methods Mol Biol 1097:491-521; Chen Y, Varani G (2013) Engineering RNA-binding proteins for biology. FEBS J 280(16):3734-3754]. It has been proposed that proteins such as the C. elegans Puf [Campbell Z T, Valley C T, Wickens M (2014) A protein-RNA specificity code enables targeted activation of an endogenous human transcript. Nat Struct Mol Biol 21(8):732-738], the human PumHD [Abil Z, Denard C A, Zhao H (2014) Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA. J Biol Eng 8(1):7], or members of the pentatricopeptide family [Coquille S, et al. (2014) An artificial PPR scaffold for programmable RNA recognition. Nat Commun 5:5729] could serve such a purpose. Each of these proteins is made of many similar units, each of which binds one RNA base.
The most extensively studied protein architecture, in the context of prospective universal single stranded RNA targeting in mammalian cells, is the human Pumilio homology domain (PumHD) [Filipovska A, Rackham 0 (2012) Modular recognition of nucleic acids by PUF, TALE and PPR proteins. Mol Biosyst 8(3):699-708; Moore F L, et al. (2003) Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins. Proc Natl Acad Sci USA 100(2):538-43; Lunde B M, Moore C, Varani G (2007) RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8(6):479-90; Wickens M, Bernstein D S, Kimble J, Parker R (2002) A PUF family portrait: 3′UTR regulation as a way of life. Trends Genet 18(3):150-157]. PumHD is a protein of 10 units, of which 8 units bind to the bases of an 8-nucleobase target RNA sequence, called the Nanos Response Element (NRE), in the reverse orientation 3′ AUAUAUGU 5′ [Spassov D S, Jurecic R (2002) Cloning and comparative sequence analysis of PUM1 and PUM2 genes, human members of the Pumilio family of RNA-binding proteins. Gene 299(1-2):195-204; Wang X, Zamore P D, Hall T M T, Tanaka Hall T M (2001) Crystal structure of a Pumilio homology domain. Mol Cell 7(4):855-865; Wang X, McLachlan J, Zamore P D, Hall T M T (2002) Modular Recognition of RNA by a Human Pumilio-Homology Domain. Cell 110(4):501-512; Cheong C-G, Hall T M T (2006) Engineering RNA sequence specificity of Pumilio repeats. Proc Natl Acad Sci USA 103(37):13635-13639; Zamore P D, Williamson J R, Lehmann R (1997) The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. RNA 3(12):1421-33; Miller M T, Higgin J J, Tanaka Hall T M, Hall T M T (2008) Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p. Nat Struct Mol Biol 15(4):397-402; Qiu C, et al. (2012) Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket. J Biol Chem 287(9):6949-57]. X-ray structures of the PumHD-NRE complex indicate that three key amino acids interact with each RNA nucleobase [Wang X, Zamore P D, Hall T M T, Tanaka Hall T M (2001) Crystal structure of a Pumilio homology domain. Mol Cell 7(4):855-865; Chen Y, Varani G (2011) Finding the missing code of RNA recognition by PUF proteins. Chem Biol 18(7):821-3].
A number of pioneering studies have shown that modifications of the wild-type PumHD can indeed bind to many sequences other than the NRE, strongly pointing towards the modularity of PumHD (the shorthand ‘Pum’ is used herein to denote any protein homologous to or derived from PumHD). Given the rich set of previous findings related to Pum proteins, it would be useful to devise a set of four canonical protein modules, each of which targets one RNA base with high specificity and fidelity, and which could be concatenated in chains of varying composition and length so as to bind desired target RNAs. A similar protein architecture, the TAL effector, has been rendered in this single-module form and has proven to be useful for targeting DNA because of its modularity [Miller J C, et al. (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143-8; Sander J D, et al. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29(8):697-8]). There are four canonical TALE protein modules, each of which targets one DNA base with high specificity and fidelity. If analogous Pum modules could be developed, they could be easily designed and used: simply concatenate a chain of modules according to the sequence of a natural target RNA, and then the protein (perhaps equipped with various reporters and effectors) could be targeted to a desired RNA.
Previous works have demonstrated, using proteins that bind to specific RNA sequences, the measurement of mRNA expression level [Ozawa T, Natori Y, Sato M, Umezawa Y (2007) Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat Methods 4(5):413-419; Yamada T, Yoshimura H, Inaguma A, Ozawa T (2011) Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes. Anal Chem 83(14):5708-5714], imaging of mRNA dynamics [Ozawa T, Natori Y, Sato M, Umezawa Y (2007) Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat Methods 4(5):413-419. Yamada T, Yoshimura H, Inaguma A, Ozawa T (2011) Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes. Anal Chem 83(14):5708-5714; Yoshimura H, Inaguma A, Yamada T, Ozawa T (2012) Fluorescent probes for imaging endogenous ??-actin mRNA in living cells using fluorescent protein-tagged pumilio. ACS Chem Biol 7(6):999-1005; Tilsner J, et al. (2009) Live-cell imaging of viral RNA genomes using a Pumilio-based reporter. Plant J 57(4):758-770; Tilsner J (2015) Pumilio-based RNA in vivo imaging. Methods Mol Biol 1217:295-328], and enhancement and suppression of mRNA translation [Campbell Z T, Valley C T, Wickens M (2014) A protein-RNA specificity code enables targeted activation of an endogenous human transcript. Nat Struct Mol Biol 21(8):732-738; Cao J, et al. (2013) Light-inducible activation of target mRNA translation in mammalian cells. Chem Commun (Camb) 49(75):8338-40; Cao J, Arha M, Sudrik C, Schaffer D V., Kane R S (2014) Bidirectional regulation of mRNA translation in mammalian cells by using PUF domains. Angew Chemie—Int Ed 53(19):4900-4904; Choudhury R, Tsai Y S, Dominguez D, Wang Y, Wang Z (2012) Engineering RNA endonucleases with customized sequence specificities. Nat Commun 3:1147].